Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

Summary

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

Abstract

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

Introduction

In the human brain, microglia are the primary innate immune cells¹. Brain development is regulated by microglia via two routes: release of diffusible factors and phagocytosis¹. Microglia-derived diffusible factors help support myelination, neurogenesis, synaptic formation, maturation, cell death, and cell survival¹. Microglia also phagocytize various elements in brain synapses, axons and in both living and dead cells^2,3,4,5,6,

Protocol

The human iPSCs used in this study were reprogrammed from fibroblasts that had been obtained through informed consent from healthy control subjects, with approval from the institutional review board (IRB). The reprogramming and characterization of iPSCs used in this study (ML15, ML27, ML40, ML56, ML141, ML 250, ML292) were described in a prior study⁵¹.

1. Maintenance of iPSCs

1. Prepare a 1:50 dilution of LDEV-free reduced growth factor basement membrane matrix.......

Discussion

The development of differentiation methods along different trajectories for pluripotent stem cells have opened many avenues for the investigation of brain function and disease processes^53,54,55. Initial studies had focused on the development of specific neuronal cell types hypothesized to be important in specific brain disorders^56,57. Recently, brain organoids have also .......

Materials

Name	Company	Catalog Number	Comments
Accutase	Sigma-Aldrich	A6964
B-27 supplement	Gibco	17504044
b-FGF	Peprotech	100-18B
BMP-4	Peprotech	120-05ET
Brainphys	StemCell Technologies	5790
CD11C antibody	Biolegend	337207	Dilution 1:200
Costar Flat Bottom Cell Culture Plates	Corning	07-200-83
Ctip2 antibody	Abcam	ab18465
CUTL1 monoclonal antibody	Abnova	H00001523-M01
DMEM/F-12, no phenol red	Gibco	21041025
dorsomorphin	Sigma-Aldrich	P5499
DPBS, no calcium, no magnesium	Gibco	14190144
Dulbecco's Modified Eagle Medium (DMEM)	Sigma-Aldrich	D6421
EasYFlask Cell Culture Flasks	Nunc	156499
Fisherbrand Cell Lifters	Fisher Scientific	08-100-240
Flt3-Ligand	Peprotech	300-19
Fluo4-AM	Life Technologies	F-14201
Geltrex LDEV Free RGF BME 1 ML	ThermoFisher Scientific	A1413201
Glutamax	ThermoFisher Scientific	35050061
GM-CSF	Peprotech	300-03
Goat Anti Chicken- IgG H&L (Alexa Fluor 488)	Abcam	ab150169	Dilution 1:1000
Goat Anti mouse- IgG H&L (Alexa Fluor 568)	Invitrogen	A-11004	Dilution 1:1000
Goat Anti Rat- IgG H&L (Alexa Fluor 405)	Abcam	ab175670	Dilution 1:1000
Goat Anti-Guinea pig IgG H&L (Alexa Fluor 405)	Abcam	ab175678	Dilution 1:1000
Goat Serum	Sigma-Aldrich	G9023
HBSS	Invitrogen	14170120
IBA1 antibody	Abcam	ab5076	Dilution 1:500
IL-34	Peprotech	200-34
INF-y	Peprotech	300-02
KiCqStart SYBR Green Primers	Sigma-Aldrich	KSPQ12012
Laminin	Sigma-Aldrich	L2020
LDN193189	Sigma-Aldrich	SML0599
Live Cell Imaging Solution	Invitrogen	A14291DJ
MAP2 antibody	Synaptic Systems	188 004
M-CSF	Peprotech	300-25
N-2 supplement	Gibco	17502001
Neurobasal medium	Life Technologies	21103049
NutriStem hPSC XF Medium	Biological Industries	01-0005
P2RY12 antibody	Biolegend	848002
Paraformaldehyde 16%	Fisher Scientific	50-980-488
Penicillin-streptomycin	Gibco	15140122
Poly-L-Orthinine	Sigma-Aldrich	P3655
SATB2 antibody	Abcam	ab51502
SB431542	Sigma-Aldrich	S4317
SCF	Stemcell Technologies	78062
SensoPlate 24-Well Glass-Bottom Plate	Greiner-Bio	662892
StemPro-34 SFM (1X)	Gibco	10639011
TMEM119 antibody	Abcam	ab185333	Dilution 1:1000
TPO	Peprotech	300-18
Triton-X	Sigma-Aldrich	9002-93-1
VEGF	Peprotech	100-20
Versene	ThermoFisher Scientific	15040066
Y-27632 dihydrochloride (ROCK inhibitor)	Tocris	1254